葡萄糖耐量因子对Hep-G2细胞葡萄糖代谢的影响

发布时间：2018-06-28 13:34

本文选题：葡萄糖耐量因子 + YSI-3.7酵母菌株　；参考：《天津科技大学》2017年硕士论文

【摘要】：铬是人体必需的微量元素,对人体糖脂代谢具有重要的作用。糖尿病患者及老年人通过适量补铬(Cr3+),可显著恢复糖耐量损伤状况,维持体内糖代谢平衡。从酵母菌中提取纯化出的天然葡萄糖耐量因子(Glucose tolerance factor,GTF),其活性中心也为Cr3+。因此,本文通过提取纯化GTF,并将其应用于胰岛素抵抗型Hep-G2细胞模型中,研究GTF结构及其对细胞糖脂代谢的作用。全文旨在建立优良、高效的GTF纯化方法,得到高产、高纯度的GTF物质,同时探究GTF在降糖降脂功能上的作用。首先,本文通过铬平板筛选出最高可在铬离子浓度为1000 μg/mL平板上生长的耐铬啤酒酵母菌YSI-3.7,将其接种于含铬发酵液中,并逐步增加培养基中铬离子浓度。结果表明,当发酵液中铬添加量达到500 μg/mL时,测得其有机铬积累最多,达到1818μg/g,且有机铬百分含量达到了 90.34%。同时,将富铬驯化得到的高产GTF酵母菌株分别进行不同规模的发酵实验,包括摇瓶发酵、15 L发酵、50 L发酵以及500 L发酵,通过测定其有机铬的含量发现,有机铬含量显著下降,由摇瓶发酵的2160μg/mL下降至500L中试发酵的765 μg/mL;有机铬占总铬含量的百分比也由87.17%下降至61.55%;菌体干生物量由0.89g/100mL到1.01g/100mL,反而有所增加。总体来看,随着发酵规模的扩大,酵母菌中有机铬和总铬含量呈下降趋势。其次,将制备得到的高产GTF的酵母菌粉经氨水提取,30%乙醇、0℃冰浴沉淀20 min,离心取上清液,测得其铬含量与蛋白含量的比值为5.02,相比单独使用氨水提取得到的0.818, GTF的提取率提高了六倍以上。将乙醇沉淀后的粗提物依次经过SuperdexTM75和Sephadex G25凝胶层析分离,得到GTF纯化物质,之后对其进行高效液相色谱(High performance liquid chromatography,HPLC)分析。结果表明,经过氨水提取,乙醇沉淀及两级柱层析纯化分离后,可得到纯度较高的GTF物质。同时,本文通过HPLC及核磁共振(Nuclear magnetic resonance,NMR)实验对GTF结构进行分析,得出谷氨酸和天门冬氨酸为GTF的主要氨基酸成分。采用HPLC和电感耦合等离子体质谱(Inductively coupled plasma - Mass spectrometry,ICP-MS )技术联用对GTF中铬价态进行分析,得出GTF中只存在Cr3+。最后,本文还采用浓度为10-6mol/L胰岛素作用Hep-G2细胞48 h,建立胰岛素抵抗细胞模型,此细胞模型可在48h内稳定维持胰岛素抵抗特性。建模成功后,将GTF及其他铬制剂应用于模型细胞中,通过测定细胞葡萄糖消耗量、甘油三酯含量(Triglyceride,TG)及游离脂肪酸含量(Non-esterified fatty acid,NEFA),对细胞的糖脂代谢作用进行评价,得出GTF铬含量在1.0 μg/mL,作用24 h时,对细胞葡萄糖代谢的促进作用最明显,葡萄糖消耗量达到18.07mmol/L,和对照组相比增加了 5%;同时,还将铬含量为1.0 μg/mL的GTF及其他铬制剂共同作用于胰岛素抵抗模型细胞中,结果显示,铬制剂对细胞糖脂代谢均有促进作用,其中GTF作用最显著(P 0.01 )。综上所述,本文通过富铬驯化,筛选得到高耐铬啤酒酵母菌株YSI-3.7,经过氨水提取、30%乙醇沉淀、两级层析柱分离,提取纯化GTF,通过氨基酸组成分析及NMR试验得出天门冬氨酸和谷氨酸为GTF主要成分;HPLC-ICP-MS技术联用得出GTF中只存在Cr3+一种铬价态;采用高胰岛素作用Hep-G2细胞建立胰岛素抵抗模型,将GTF及其它铬制剂分别作用于模型细胞中,研究分析其对细胞糖脂代谢的作用。
[Abstract]:Chromium is an essential trace element in human body, which plays an important role in the metabolism of glycolipid in the human body. By using a proper amount of chromium supplementation (Cr3+), the diabetic patients and the elderly can significantly restore the impaired glucose tolerance and maintain the balance of glucose metabolism in the body. The natural glucose tolerance factor (Glucose tolerance factor, GTF) extracted from the yeast and its activity The heart also is Cr3+., therefore, the purpose of this paper is to extract and purify GTF and apply it to the insulin resistance Hep-G2 cell model to study the structure of GTF and its effect on the metabolism of glycemic cells. The full text aims to establish an excellent and efficient GTF purification method, to obtain high yield and high purity GTF substances, and to explore the role of GTF in reducing the function of glucose lowering and lipid lowering. A chromium resistant Saccharomyces cerevisiae YSI-3.7, which can grow on the 1000 g/mL plate of chromium ion concentration, was screened by chromium plate, and inoculated into the chromium containing fermented liquid and gradually increased the concentration of chromium ion in the medium. The results showed that the accumulation of chromium in the fermentation broth was up to 500 g /mL, and the accumulation of organic chromium was up to 1818 mu. G/g, and the content of organic chromium was 90.34%., and the high yield GTF yeast strains were fermented at different sizes, including shake flask fermentation, 15 L fermentation, 50 L fermentation and 500 L fermentation. By measuring the content of organic chromium, the content of organic chromium decreased significantly, and 2160 g/mL decreased from shake flask fermentation. The 765 micron g/mL was fermented in 500L, and the percentage of organic chromium in the total chromium content decreased from 87.17% to 61.55%; the dry biomass of the bacteria increased from 0.89g/100mL to 1.01g/100mL. As a whole, the content of chromium and total chromium in the yeast decreased with the expansion of the fermentation scale. Secondly, the high yield GTF yeast was prepared. The bacteria powder was extracted by ammonia water, 30% ethanol and 20 min in 0 centigrade ice bath. The ratio of chromium content to protein content was 5.02, and the extraction rate of GTF was six times higher than that obtained by the extraction of ammonia water alone. The crude extract after ethanol precipitation was separated by SuperdexTM75 and Sephadex G25 gel chromatography in turn. After purifying the substance to GTF, the high performance liquid chromatography (High performance liquid chromatography, HPLC) was analyzed. The results showed that the purity of GTF substance could be obtained after the extraction of ammonia, the ethanol precipitation and the purification and separation of the two column chromatography. At the same time, the experiment was carried out by HPLC and nuclear magnetic resonance (Nuclear magnetic resonance, NMR). The TF structure is analyzed and the main amino acid components of GTF are glutamic acid and aspartic acid. HPLC and inductively coupled plasma mass spectrometry (Inductively coupled plasma Mass spectrometry, ICP-MS) are used to analyze the valence state of chromium in GTF. It is concluded that GTF is in the last of Cr3+., and the concentration is also used as the islet. Hep-G2 cells were treated with 48 h to establish an insulin resistance cell model. This cell model could maintain insulin resistance stability in 48h. After the modeling, GTF and other chromium preparations were applied to the model cells, and the glucose consumption, triglyceride content (Triglyceride, TG) and free fatty acid content (Non-esterified f) were measured. Atty acid, NEFA), to evaluate the glycolipid metabolism of cells, it is concluded that the content of GTF chromium is 1 u g/mL, when the action of 24 h, the promotion of glucose metabolism is the most obvious, the consumption of glucose reaches 18.07mmol/L, which is increased by 5% compared with the control group; meanwhile, the chromium content of 1 u g/mL and other chromium preparations are combined in the pancreas. In the island element resistance model cells, the results showed that the chromium preparation had the promotion effect on the cell glycolipid metabolites, and the GTF effect was most significant (P 0.01). In summary, the high chromium resistant Saccharomyces cerevisiae strain YSI-3.7 was screened through the acclimation of chromium rich, through the ammonia extraction, the 30% ethanol precipitation, the two level chromatography column separation, the extraction and purification of GTF, through the amino acid extraction. The composition analysis and NMR test showed that aspartic acid and glutamic acid were the main components of GTF; HPLC-ICP-MS was used to conclude that only a chromium valence state in Cr3+ was found in GTF; the insulin resistance model was established by high insulin action Hep-G2 cells, and GTF and other chromium preparations were used in the model cells respectively, and the metabolism of cell glycolipid was studied and analyzed. Effect.
【学位授予单位】：天津科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R587.1

【参考文献】